Bromodomain extra terminal (BET) proteins are critical readers of the histone acetyl marks, which generally signal active transcription. Human BET proteins include the ubiquitous BRD2, BRD3, and BRD4, as well as the testis-specific BRDt. They are key regulators of mitosis and pluripotency reprogramming among many other cellular and biological processes including transcription, cell cycle, transcriptional memory, and stem cell identity, as well as viral replication and transcription. Bromodomains (BD) in BET proteins are the acetyllysine(KAc)-binding modules capable of binding to acetylated core histones. BET proteins are characterized by two tandem bromodomains (BD-I and BD-II) at their N-termini. Of note, the two tandem BDs within each BET member are much more diverse than the equivalent BDs among BET member proteins. In spite of intense investigation, many fundamental questions about these readers of chromatin acetyl marks remain unsolved. Why are there two BDs within each BET protein? Why are the two tandem BDs of each BET protein more diverse than the equivalent BDs among the BET member proteins? What are the specific acetyl marks of histones each individual BD differentially recognizes? Does each individual BD specifically regulate any biological process? Why do each of human BRD3 and BRD4 has both long and short isoforms? How is the chromatin-reading activity of each BD regulated? As mitotic proteins, how is their mitotic activity tightly controlled? Recent findings in my laboratory have paved an avenue to answering many, if not all, of these fundamental questions. We recently reported that BRD3R, a truncated isoform of human BRD3, uniquely enhances cellular pluripotency reprogramming via regulating mitosis. Our further preliminary data suggest that these BRD3R activities are a result of unmasking of the chromatin-reading activities of the second bromodomain (BD-II) of BRD3/BRD3R due to the absence of the long C-terminal tail. Our additional data suggest that intramolecular masking of the BD-II activities may be a general mechanism among BET proteins. We will study how intramolecular masking/unmasking regulate biological and chemical functions of the two distinct BET bromodomains (Aims 1, 2, and 3). We will also identify the chromatin underpinnings behind the different biological and cellular functions of the two distinct KAc-binding modules of BET proteins (Aim 3). To achieve our goals, we will use up-to-date yet firmly established approaches including the chemically defined reprogramming media, ChIP-seq, ChIP-qPCR, RNA-seq, bioinformatics, histone-tail-peptide pull-down, histone-peptide array, ligand affinity assay, mutagenesis, protein-protein interaction, and others. Dysregulation of the BET KAc-reading activities are implicated in various human diseases such as inflammation, diabetes, and various cancers. The fundamental knowledge gained in this proposed study will provide a scientific basis for the disease control and management for the BET-associated diseases.